Abstract: The accurate regulation of gene expression is crucial for proper development of multi-cellular organisms and for maintaining cell homeostasis in adult tissues. De-regulation of gene expression is the hallmark of an ever-increasing number of human diseases and many drugs directly target key gene transcription factors. Current therapies however affect transcription on a pan-genomic scale, without specificity and without the ability to fine-tune gene activity. Our lack of understanding of the detailed pathways underlying transcriptional regulation precludes designing targeted therapies. Moreover, current experimental tools cannot address the complexity for the biochemical system involved in human mRNA transcription, comprised by multi-component core transcription machinery and multiple additional layers of regulatory factors, co-activators, repressors and chromatin remodeling complexes. Critically, no current experimental method can observe the dynamics of these factors inside a live cell, dissect transcription kinetics, identify rate-limiting steps that are subject to regulation, and ultimately uncover mechanistic principles. My goal is to develop the enabling optical imaging and spectroscopy tools that can provide high-resolution, dynamic data, to facilitate describing transcription regulation in detailed molecular terms. During the period of this award I envision accomplishing the following towards this direction: (1) create innovating technologies that will make possible to visualize complex biochemical processes at the single-molecule level, in real-time, inside living cells; (2) discern in vivo mechanisms by which transcription factors regulate rate-limiting steps in the cycle of mRNA synthesis; (3) provide a framework for understanding signal integration and combinatorial control of gene expression. The proposed research builds upon my unique expertise at the border of physical and biological sciences, and seeks to create a new synthesis of ideas and methodologies towards novel strategies to understand and control gene expression, in a way relevant to medicine. Public Health Relevance: Transcription is the first and most highly regulated step in gene expression. Aberrant transcription is associated with an ever-growing number of diseases and several drugs directly target transcription factors. This project aims at elucidating the function f the core molecular transcription machinery and provide new strategies for controlling its regulation for therapeutic applications.